Transport of chimeric proteins that contain a carboxy-terminal targeting signal into plant microbodies

Malate synthase is a glyoxysome-specific enzyme. The carboxy-terminal tripeptide of the enzyme is Ser—Arg—Leu (SRL), which is known to function as a peroxisomal targeting signal in mammalian cells. To analyze the function of the carboxy-terminal amino acids of pumpkin malate synthase in plant cells, a chimeric gene was constructed that encoded a fusion protein which consisted of β-glucuronidase and the carboxyl terminus of the enzyme. The fusion protein was expressed and accumulated in transgenic Arabidopsis that had been transformed with the chimeric gene. Immunocytochemical analysis of the transgenic plants revealed that the carboxy-terminal five amino acids of pumpkin malate synthase were sufficient for transport of the fusion protein into glyoxysomes in etiolated cotyledons, into leaf peroxisomes in green cotyledons and in mature leaves, and into unspecialized microbodies in roots, although the fusion protein was no longer transported into microbodies when SRL at the carboxyl terminus was deleted. Transport of proteins into glyoxysomes and leaf peroxisomes was also observed when the carboxy-terminal amino acids of the fusion protein were changed from SRL to SKL, SRM, ARL or PRL. The results suggest that tripeprides with S, A or P at the −3 position, K or R at the −2 position, and L or M at the carboxyl terminal position can function as a targeting signal for three kinds of plant microbody.     

Identification of amino acids in the gamma-carboxylation recognition site on the propeptide of prothrombin

A gamma-carboxylation recognition site on the propeptide of the vitamin K-dependent blood coagulation proteins directs the carboxylation of glutamic acid residues by binding to the vitamin K-dependent carboxylase. To determine residues that define this site, we evaluated the effect of mutation of certain residues in the prothrombin propeptide on the extent of carboxylation. The prothrombin cDNA modified by site-specific mutagenesis was expressed in Chinese hamster ovary cells using a system that yields functional fully carboxylated prothrombin. The cell supernatants containing recombinant prothrombin were evaluated for the extent of gamma-carboxylation by immunoassay. Conformation-specific anti-prothrombin:Ca(II)-specific antibodies measure native completely carboxylated prothrombin; anti-prothrombin:total antibodies measure all forms of prothrombin, regardless of gamma-carboxyglutamic acid content. Mutation of His-18 to Gly, Val-17 to Ser, Leu-15 to Gly or Asp, or Ala-10 to Asp was associated with a partial (30-65%) inhibition of gamma-carboxylation. Mutation of Ala-14 to Ser or Ser-8 to Val did not inhibit gamma-carboxylation. From this and earlier work, residues whose mutation leads to a significant impairment of carboxylation include His-18, Val-17, Phe-16, Leu-15, and Ala-10. Residues whose mutation does not alter the carboxylation recognition site include Ala-14, Ser-8, Arg-4, and Arg-1. To determine the size of the recognition site, the in vitro carboxylation of propeptide-containing synthetic peptides was compared. A 28-residue peptide, based upon residues -18 to +10 of prothrombin, and a 54-residue peptide, based upon residues -18 to +36 of prothrombin, were carboxylated by partially purified bovine carboxylase with similar Km values of 2-5 microM. These results indicate that the gamma-carboxyglutamic acid-rich region of prothrombin makes a minimal contribution to carboxylase binding. A molecular surface of about five amino acids located within the propeptide appears to define the carboxylation recognition site on the precursor forms of the vitamin K-dependent proteins.     

The transmembrane domain directs TLR9 to intracellular compartments that contain TLR3

Toll-like receptors (TLRs) are type I transmembrane (TM) proteins indispensable for sensing microbial and viral infection. Despite their conserved primary structures, some TLRs that detect pathogen-derived nucleic acids (TLR3, TLR7, TLR8, and TLR9) are retained in the cytoplasm. The intracellular localization of TLR9 is important for its ability to discriminate self- and non-self DNA, but the mechanism by which it is retained in the cytoplasm is unclear. In the present study, we found that the TM domain of TLR9 directs its intracellular localization. The TM domain of TLR9 also targets CD25, a heterologous type I TM protein, to intracellular compartments that contain TLR9. We also found that TLR9 generally co-localizes with TLR3, although its linker region, not its TM domain, directs intracellular localization of TLR3. These data demonstrate that the TM domain of TLR9 is a critical regulatory element that targets TLR9 to its intracellular location.     

Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site

Synthetic peptides including the gamma-carboxylation recognition site and acidic amino acids were compared as substrates for vitamin K-dependent gamma-carboxylation by bovine liver carboxylase. The 28-residue proPT28 (proprothrombin -18 to +10) and proFIX28 (pro-Factor IX -18 to +10) were carboxylated with a Km of 3 microM. The Vmax of proPT28 was 2-3 times greater than that of proFIX28. An analog of proFIX28 that contained the prothrombin propeptide had a Vmax 2-3-fold greater than an analog of proPT28 that contained the Factor IX propeptide. proFIX28/RS-1, based upon Factor IX Cambridge, proFIX28/RQ-4, based upon Factor IX Oxford 3, and proFIX28 had equivalent Km and Vmax values. Analogs of proPT28 containing Ala6-Glu7 or Glu6-Ala7 were carboxylated at equivalent rates. A peptide containing Asp6-Asp7 was carboxylated at a rate of about 1% of that of Glu carboxylation. Carboxylation of peptides containing Asp6-Glu7 and Glu6-Asp7 yielded results identical with peptides containing Ala6-Glu7 and Glu6-Ala7. Carboxymethylcysteine was not carboxylated when substituted for Glu6 in a peptide containing Asp7. These results indicate that the prothrombin propeptide is more efficient in the carboxylation process than is the Factor IX propeptide, but that both propeptides direct carboxylation; the gamma-carboxylation recognition site does not include residues -4 and -1; aspartic acid and carboxymethylcysteine are poor substrates for the carboxylase, but aspartic acid does not inhibit the carboxylation of adjacent glutamic acids.     

Structure of the propeptide of prothrombin containing the gamma-carboxylation recognition site determined by two-dimensional NMR spectroscopy

The propeptides of the vitamin K dependent blood clotting and regulatory proteins contain a gamma-carboxylation recognition site that directs precursor forms of these proteins for posttranslational gamma-carboxylation. Peptides corresponding to the propeptide of prothrombin were synthesized and examined by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). CD spectra indicate that these peptides have little or no secondary structure in aqueous solutions but that the addition of trifluoroethanol induces or stabilizes a structure containing alpha-helical character. The maximum helical content occurs at 35-40% trifluoroethanol. This trifluoroethanol-stabilized structure was solved by two-dimensional NMR spectroscopy. The NMR results demonstrate that residues -13 to -3 form an amphipathic alpha-helix. NMR spectra indicate that a similar structure is present at 5 degrees C, in the absence of trifluoroethanol. Of the residues previously implicated in defining the gamma-carboxylation recognition site, four residues (-18, -17, -16, and -15) are adjacent to the helical region and one residue (-10) is located within the helix. The potential role of the amphipathic alpha-helix in the gamma-carboxylation recognition site is discussed.     

RsrII--a novel restriction endonuclease with a heptanucleotide recognition site.

A sequence-specific endonuclease present in extracts of Rhodopseudomonas sphaeroides 630 has been purified and characterized. The enzyme, Rsr II, recognises and cleaves the palindromic heptanucleotide sequence: (sequence; see test) By virtue of its unusual specificity, RsrII cuts most DNA molecules very infrequently which should facilitate the physical mapping of large genomes.     

Binding matrix: a novel approach for binding site recognition

Recognition of protein-DNA binding sites in genomic sequences is a crucial step for discovering biological functions of genomic sequences. Explosive growth in availability of sequence information has resulted in a demand for binding site detection methods with high specificity. The motivation of the work presented here is to address this demand by a systematic approach based on Maximum Likelihood Estimation. A general framework is developed in which a large class of binding site detection methods can be described in a uniform and consistent way. Protein-DNA binding is determined by binding energy, which is an approximately linear function within the space of sequence words. All matrix based binding word detectors can be regarded as different linear classifiers which attempt to estimate the linear separation implied by the binding energy function. The standard approaches of consensus sequences and profile matrices are described using this framework. A maximum likelihood approach for determining this linear separation leads to a novel matrix type, called the binding matrix. The binding matrix is the most specific matrix based classifier which is consistent with the input set of known binding words. It achieves significant improvements in specificity compared to other matrices. This is demonstrated using 95 sets of experimentally determined binding words provided by the TRANSFAC database.     

Pannexin1 channels contain a glycosylation site that targets the hexamer to the plasma membrane

Pannexins are newly discovered channel proteins expressed in many different tissues and abundantly in the vertebrate central nervous system. Based on membrane topology, folding and secondary structure prediction, pannexins are proposed to form gap junction-like structures. We show here that Pannexin1 forms a hexameric channel and reaches the cell surface but, unlike connexins, is N-glycosylated. Using site-directed mutagenesis we analyzed three putative N-linked glycosylation sites and examined the effects of each mutation on channel expression. We show for the first time that Pannexin1 is glycosylated at Asn-254 and that this residue is important for plasma membrane targeting. The glycosylation of Pannexin1 at its extracellular surface makes it unlikely that two oligomers could dock to form an intercellular channel. Ultrastructural analysis by electron microscopy confirmed that Pannexin1 junctional areas do not appear as canonical gap junctions. Rather, Pannexin1 channels are distributed throughout the plasma membrane. We propose that N-glycosylation of Pannexin1 could be a significant mechanism for regulating the trafficking of these membrane proteins to the cell surface in different tissues.     

The carboxy-terminal end of glycolate oxidase directs a foreign protein into tobacco leaf peroxisomes.

The carboxy-terminal residues of several peroxisomal proteins were shown to act as a peroxisomal targetting signal. This study was conducted to test whether the C-terminus of glycolate oxidase, a key enzyme in the glycolate metabolism pathway, is functioning as a targetting signal that directs proteins into plant leaf peroxisomes. A chimeric gene coding for a fusion protein composed of the C-terminal-truncated beta-glucuronidase, a synthetic linker of four amino acids and the last six C-terminal amino acids of glycolate oxidase, was constructed. Transformation of tobacco plants with the chimeric gene resulted in expression of beta-glucuronidase enzymic activity. About 50% of the transgenic beta-glucuronidase activity was localized to the peroxisomes. The results indicate that the six C-terminal amino acid residues of glycolate oxidase act as a targetting signal that is recognized by leaf peroxisomes.     

Identification of a novel domain of Ras and Rap1 that directs their differential subcellular localizations

The small GTPase Ha-Ras and Rap1A exhibit high mutual sequence homology and share various target proteins. However, they exert distinct biological functions and exhibit differential subcellular localizations; Rap1A is predominantly localized in the perinuclear region including the Golgi apparatus and endosomes, whereas Ha-Ras is predominantly localized in the plasma membrane. Here, we have identified a small region in Rap1A that is crucial for its perinuclear localization. Analysis of a series of Ha-Ras-Rap1A chimeras shows that Ha-Ras carrying a replacement of amino acids 46-101 with that of Rap1 exhibits the perinuclear localization. Subsequent mutational studies indicate that Rap1A-type substitutions within five amino acids at positions 85-89 of Ha-Ras, such as NNTKS85-89TAQST, NN85-86TA, and TKS87-89QST, are sufficient to induce the perinuclear localization of Ha-Ras. In contrast, substitutions of residues surrounding this region, such as FAI82-84YSI and FEDI90-93FNDL, have no effect on the plasma membrane localization of Ha-Ras. A chimeric construct consisting of amino acids 1-134 of Rap1A and 134-189 of Ha-Ras, which harbors both the palmitoylation and farnesylation sites of Ha-Ras, exhibits the perinuclear localization like Rap1A. Introduction of a Ha-Ras-type substitution into amino acids 85-89 (TAQST85-89NNTKS) of this chimeric construct causes alteration of its predominant subcellular localization site from the perinuclear region to the plasma membrane. These results indicate that a previously uncharacterized domain spanning amino acids 85-89 of Rap1A plays a pivotal role in its perinuclear localization. Moreover, this domain acts dominantly over COOH-terminal lipid modification of Ha-Ras, which has been considered to be essential and sufficient for the plasma membrane localization.     

Capnocytophaga spp. contain sulfonolipids that are novel in procaryotes.

A group of unusual sulfonolipids was found in bacteria of the genus Capnocytophaga. One of these lipids, to which we have assigned the trivial name capnine, was isolated in 98% pure form and was identified, by infrared absorption spectrometry, high-resolution mass spectrometry, and other methods, as 2-amino-3-hydroxy-15-methylhexadecane-1-sulfonic acid. Another lipid appears to be an N-acylated version of capnine; after acid hydrolysis, its sulfur was recovered in a form chromatographically indistinguishable from that of capnine. The new lipids are related structurally to sphingosine and the ceramides, respectively, but differ markedly from those compounds in important respects, notably the presence of the sulfonate group. Some Capnocytophaga strains accumulated mostly capnine, whereas others accumulated mostly N-acylcapnine. All seven strains examined were found to contain the new lipids, in amounts ranging from 7 to 16 mumol/g of cells (wet weight). The lipids were found in isolated cell envelopes, where they were present in amounts ranging up to 400 mg/g of envelope protein; they are, accordingly, major cell components.     

Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants

A novel, constitutively expressed gene, designated MtHP, was isolated from the model legume species Medicago truncatula. Sequence analysis indicates that MtHP most likely belongs to the PR10 multi-gene family. The MtHP promoter was fused to a -glucuronidase gene to characterize its expression in different plant species. Transient assay by microprojectile bombardment and hairy root transformation by Agrobacterium rhizogenes revealed GUS expression in leaf, stem, radicle and root in M. truncatula. Detailed analysis in transgenic Arabidopsis plants demonstrated that the promoter could direct transgene expression in different tissues and organs at various developmental stages; its expression pattern was similar to that of CaMV35S promoter, and the level of expression was higher than the reporter gene driven by CaMV35S promoter. Deletion analysis revealed that even a 107 bp fragment of the promoter could still lead to a moderate level of expression. The promoter was further characterized in white clover (Trifolium repens), a widely grown forage legume species. Strong constitutive expression was observed in transgenic white clover plants. Compared with CaMV35S promoter, the level of GUS activity in transgenic white clover was higher when the transgene was driven by MtHP promoter. Thus, the promoter provides a useful alternative to the CaMV35S promoter in plant transformation for high levels of constitutive expression.     

Characterization of novel M-superfamily conotoxins with new disulfide linkage

The M-superfamily with the typical Cys framework (-CC-C-C-CC-) is one of the seven major superfamilies of conotoxins found in the venom of cone snails. Based on the number of residues in the last Cys loop (between C4 and C5), M-superfamily conotoxins can be provisionally categorized into four branches (M-1, M-2, M-3, M-4) [Corpuz GP, Jacobsen RB, Jimenez EC, Watkins M, Walker C, Colledge C, Garrett JE, McDougal O, Li W, Gray WR, et al. (2005) Biochemistry44, 8176-8186]. Here we report the purification of seven M-superfamily conotoxins from Conus marmoreus (five are novel and two are known as mr3a and mr3b) and one known M-1 toxin tx3a from Conus textile. In addition, six novel cDNA sequences of M-superfamily conotoxins have been identified from C. marmoreus, Conus leopardus and Conus quercinus. Most of the above novel conotoxins belong to M-1 and M-2 and only one to M-3. The disulfide analyses of two M-1 conotoxins, mr3e and tx3a, revealed that they possess a new disulfide bond arrangement (C1-C5, C2-C4, C3-C6) which is different from those of the M-4 branch (C1-C4, C2-C5, C3-C6) and M-2 branch (C1-C6, C2-C4, C3-C5). This newly characterized disulfide connectivity was confirmed by comparing the HPLC profiles of native mr3e and its two regioselectively folded isoforms. This is the first report of three different patterns of disulfide connectivity in conotoxins with the same cysteine framework.     

A novel inhibitor of farnesyltransferase with a zinc site recognition moiety and a farnesyl group

Protein prenylation such as farnesylation and geranylgeranylation is associated with various diseases. Thus, many inhibitors of prenyltransferase have been developed. We report novel inhibitors of farnesyltransferase with a zinc-site recognition moiety and a farnesyl/dodecyl group. Molecular docking analysis showed that both parts of the inhibitor fit well into the catalytic domain of farnesyltransferase. The synthesized inhibitors showed activity against farnesyltransferase in vitro and inhibited proliferation of the pancreatic cell line AsPC-1. Among the compounds with farnesyl and dodecyl groups, the inhibitor with a farnesyl group was found to have stronger and more selective activity.     

Mutants of Streptococcus pneumoniae that contain a temperature-sensitive autolysin

Two mutants of Streptococcus pneumoniae deficient in autolysin activity produced a protein that showed immunological identity with the N-acetyl-muramyl-L-alanyl-amidase present in the wild-type strain, when tested with antiserum obtained against this enzyme. The protein was produced by the mutant cultures grown either at 37 degrees C or at 30 degrees C, although only the cell extracts obtained at 30 degrees C showed significant cell wall hydrolysing activity. In contrast to the lysis resistance of these bacteria grown at 37 degrees C, mutant cultures grown at 30 degrees C exhibited significant degrees of autolysis when treated with detergent or cell wall inhibitors. Extracts of the mutant cultures contained a cell wall hydrolysing activity that was rapidly inactivated during incubation at 37 degrees C.     

Evidence that three adhesive proteins interact with a common recognition site on activated platelets

Synthetic peptides corresponding to the extreme COOH terminus of the gamma chain of fibrinogen gamma 400-411, (400)HHLGGAKQAGDV(411), have been used to analyze recognition specificities of the platelet-binding sites for fibrinogen, fibronectin, and von Willebrand factor. gamma 403-411 did not inhibit 125I-fibrinogen binding to platelets. In contrast, gamma 402-411 produced dose-dependent inhibition of fibrinogen binding to ADP and thrombin-stimulated living or fixed cells and was a competitive antagonist. Inhibitory activity was not modified by addition of one (gamma 401-411) or two (gamma 400-411) histidinyl residues to the NH2 terminus, but peptides with a trifluoroacetyl group on the epsilon-amino group of lysine 406 were inactive. 125I-Fibronectin and 125I-von Willebrand factor binding to thrombin-stimulated living or fixed cells was inhibited in the same dose range by the same set of peptides which inhibited fibrinogen binding and not by gamma 403-411 or trifluoroacetate-blocked peptides. The capacity of the peptides to inhibit binding to cells with an expressed receptor, i.e. fixed cells, excludes an effect on receptor induction. Thus, these results suggest that the three adhesive glycoproteins share a common site on thrombin-activated platelets, and a 10-amino acid peptide, corresponding to gamma 402-411, defines the recognition specificity of this site.